Targeting tumor cell-derived CCL2 as a strategy to overcome Bevacizumab resistance in ETV5+ colorectal cancer

Abstract

In our previous study, ETV5 mediated-angiogenesis was demonstrated to be dependent upon the PDGF-BB/PDGFR-β/Src/STAT3/VEGFA pathway in colorectal cancer (CRC). However, the ability of ETV5 to affect the efficacy of anti-angiogenic therapy in CRC requires further investigation. Gene set enrichment analysis (GSEA) and a series of experiments were performed to identify the critical candidate gene involved in Bevacizumab resistance. Furthermore, the ability of treatment targeting the candidate gene to enhance Bevacizumab sensitivity in vitro and in vivo was investigated. Our results revealed that ETV5 directly bound to the VEGFA promoter to promote translation of VEGFA. However, according to in vitro and in vivo experiments, ETV5 unexpectedly accelerated antiVEGF therapy (Bevacizumab) resistance. GSEA and additional assays confirmed that ETV5 could promote angiogenesis by inducing the secretion of another tumor angiogenesis factor (CCL2) in CRC cells to facilitate Bevacizumab resistance. Mechanistically, ETV5 upregulated CCL2 by activating STAT3 to facilitate binding with the CCL2 promoter. ETV5 induced-VEGFA translation and CCL2 secretion were mutually independent mechanisms, that induced angiogenesis by activating the PI3K/AKT and p38/MAPK signaling pathways in human umbilical vein endothelial cells (HUVECs). In CRC tissues, ETV5 protein levels were positively associated with CD31, CCL2, and VEGFA protein expression. CRC patients possessing high expression of ETV5/VEGFA or ETV5/CCL2 exhibited a poorer prognosis compared to that of other patients. Combined antiCCL2 and antiVEGFA (Bevacizumab) treatment could inhibit tumor angiogenesis and growth more effectively than single treatments in CRCs with high expression of ETV5 (ETV5⁺ CRCs). In conclusion, our results not only revealed ETV5 as a novel biomarker for anti-angiogenic therapy, but also indicated a potential combined therapy strategy that involved in targeting of both CCL2 and VEGFA in ETV5⁺ CRC.

Fig. 1. ETV5 induces Bevacizumab resistance in CRC in vivo.

a The CAM assay was used to examine effect of Bevacizumab and VEGFA on blood vessel formation after stimulation with the supernatants from the indicated cells. Data are presented as mean ± SD of three independent experiments. b Tumor volumes were calculated to measure tumorigenesis ability. c Images of xenografted tumors after subcutaneous injection of mice with the indicated cells (n = 5). d The average tumor weight for each group was calculated. e Representative images of HE staining and IHC staining for CD31 and VEGFA in subcutaneous tumor tissues of the RKO/Vector, RKO/Vector+Bev(2 mg/kg), RKO/ETV5, and RKO/ETV5+Bev(2 mg/kg) groups. “*” represents in comparison with the control. Bev Bevacizumab. **p < 0.01, ***p < 0.001, ****p < 0.0001. ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001.

Fig. 2. Ectopic overexpression of ETV5 promotes Bevacizumab resistance in CRC in vitro.

a CCK-8 assay was used to analyze the effect of Bevacizumab or recombinant human VEGFA protein on proliferation of HUVECs incubated with conditioned media collected from the indicated CRC cells. b Representative images of the formation of HUVEC tubules incubated with supernatants collected from the indicated cells and treatment with Bevacizumab or recombinant human VEGFA protein. Bev Bevacizumab. Data are presented as mean ± SD of three independent experiments. “*” represents in comparison with the control. **p < 0.01, ***p < 0.001, ****p < 0.0001. ^##p < 0.01, ^###p < 0.001.

Fig. 3. CCL2 is another pro-angiogenic factor that is regulated by ETV5 in CRC.

a GSEA analysis revealed pathways that were positively related to ETV5 expression in CRC according to TCGA database results. b A heatmap was used to display the significantly enriched genes in the chemokine signaling pathway. c Among the significantly enriched genes, four chemokines exhibited pro-angiogenic roles (CXCL5, CXCL11, CCL2, and CCL13). The expression of these chemokines was compared between HT29/Control and HT29/shETV5 cells (GSE112628). d qRT-PCR was used to examine the expression of CXCL5, CXCL11, CCL2, and CCL13 in HT29/shNC and HT29/shETV5 cells. e CCL2 concentrations in the supernatants of HT29/Vector, HT29/shETV5, RKO/Vector, and RKO/ETV5 cells were determined by ELISA. Data are presented as mean ± SD of three independent experiments. f Representative images of IHC staining of CCL2 in subcutaneous tumor tissues from the RKO/Vector, RKO/Vector+Bev, RKO/ETV5, and RKO/ ETV5+Bev groups. Bev Bevacizumab. “*” represents in comparison with the control. **p < 0.01, ***p < 0.001.

Fig. 4. ETV5 activates STAT3-mediated transcriptional activation of CCL2 in CRC.

a, b CRC cell lines were transfected with the indicated lentiviruses and treated with STAT3 inhibitor or STAT3 activator. Next, western blots were used to detect the expression of STAT3, pSTAT3, CCL2, and GAPDH. c, d ELISA analysis of CCL2 secretion after treatment with STAT3 inhibitor or STAT3 activator in ETV5 downregulated/overexpressing cells. e Diagram of the CCL2 promoter, where the black marker indicates the STAT3-binding sites. f ChIP was performed using an anti-STAT3 antibody in RKO cells to analyze STAT3 binding to the CCL2 promoter. qRT-PCR experiments were performed using primers against the indicated area in the CCL2 promoter, and the indicated region showed significant enrichment compared to that of the control. Data are presented as mean ± SD of three independent experiments. “*” represents in comparison with the control. ***p < 0.001, ****p < 0.0001, ^## p < 0.01, ^###p < 0.001.

Fig. 5. Tumor cell-derived CCL2 partially contributes to ETV5-mediated angiogenesis in CRC.

a ELISA analysis of CCL2 and VEGFA secretion after treatment with recombinant human VEGFA, CCL2, Bev, or anti-CCL2 in ETV5 downregulated/overexpressing cells. Bev Bevacizumab. b Representative images of the formation of HUVEC tubules following incubation with supernatants collected from the indicated cells and treatment with recombinant human CCL2 protein or CCL2 antibody. c The CAM assay was used to examine the effect of recombinant human CCL2 protein or CCL2 antibody on blood vessel formation after stimulation with the supernatants from the indicated cells. Data are presented as mean ± SD of three independent experiments. “*” represents in comparison with the control. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 6. Combination treatment using antiVEGFA and antiCCL2 synergistically inhibits tumor growth and angiogenesis in CRC.

a Representative images of the formation of HUVEC tubules following incubation with supernatants collected from the indicated cells and treatment with recombinant human VEGFA and CCL2 protein or Bev and antiCCL2. Data are presented as mean ± SD of three independent experiments. b HUVECs were incubated with the indicated supernatants and treated with recombinant human VEGFA and CCL2 protein or Bev and antiCCL2. Activation of the VEGFR downstream signaling pathway was determined according to western blotting. GAPDH was used as a loading control. c The CAM assay was used to examine effect of a combination treatment using recombinant human VEGFA and CCL2 protein or a combination of Bevacizumab and antiCCL2 on blood vessel formation after stimulation with the supernatants from the indicated cells. Data are presented as mean ± SD of three independent experiments. d Tumor volumes were calculated to measure tumorigenesis ability of RKO/ETV5 cells after receiving antiVEGFA or/and antiCCL2 treatment. e Images of xenografted tumors derived from RKO/ETV5 cells in every group. The average tumor weight for each group (n = 5) was calculated. f Representative images of HE stains and IHC staining for CD31 and ki67 in subcutaneous tumor tissue in the RKO/ETV5, RKO/ETV5+Bev, and RKO/ETV5+Bev+anti-CCL2 groups. Bev Bevacizumab. “*” represents in comparison with the control. **p < 0.01, ***p < 0.001, ****p < 0.0001. ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001.

Fig. 7. ETV5, VEGFA, and CCL2 show positive expression correlations with angiogenesis and are positively correlated with poor prognosis in CRC.

a Representative images of IHC staining for ETV5, VEGFA, CCL2, and CD31 in the 75-patient cohort. b Expression of ETV5, VEGFA, CCL2, and CD31 was up-regulated in CRC tissues compared to levels in normal tissues (all p < 0.0001). c Expression of ETV5 was positively related to expression of VEGFA (p < 0.0001), CCL2 (p = 0.0342), and CD31 (p < 0.0001) in CRC tissues. d VEGFA (p < 0.0001) and CCL2 (p = 0.0002) were significantly associated with CD31 in CRC tissues. e, f Disease-free survival (DFS) and overall survival (OS) curves of the 75 patients in the cohort, as stratified by ETV5 and VEGFA expression patterns or by ETV5 and CCL2 expression patterns. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 8. Schematic of ETV5-mediated anti-VEGF therapy resistance in ETV5⁺ CRC.

In CRC, ETV5 could promote angiogenesis via the secretion of VEGFA and CCL2. When ETV5⁺ CRC was treated with Bevacizumab, paracrine CCL2 could induce angiogenesis by activating the MAPK and AKT pathways in human umbilical vein endothelial cells, ultimately resulting in Bevacizumab resistance. Therefore, a combination using Bevacizumab and antiCCL2 treatment could synergistically suppress angiogenesis by simultaneously neutralizing ETV5-induced VEGFA and CCL2 in CRC, and this combined therapy might provide promising anti-angiogenic strategy for ETV5⁺ CRCs.